G418快速筛选酵母SMD1168表达人sCR1高拷贝克隆菌.docVIP

G418快速筛选酵母SMD1168表达人sCR1高拷贝克隆菌 周明武1 张长远2 刘亚利3 王广兰4﹡ 刘力源4 仓宝成4 裴 佩4 张凌云5 （450042 河南郑州,解放军第153中心医院全军创伤骨科中心1；神经外科2；心肾内科3；检验中心4；省军区门诊部5；） [摘要] 目的：采用酵母SMD1168细胞和分泌型载体pPIC9K，构建、表达人sCR1，用G418快速筛选及鉴定sCR1高拷贝重组转化克隆菌。方法：从人外周血中提取总RNA，经RT-PCR获得人sCR1全长cDNA，然后构建含人sCR1的重组质粒（pPIC9K-sCR1），经电转化将其克隆入酵母SMD1168细胞中，利用G418的抗性，快速筛选转化克隆菌。结果：经G418快速筛选、PCR和Western blot鉴定分析，获得了高拷贝整合阳性克隆菌，经甲醇诱导、表达、纯化后得到较纯的重组人sCR1融合蛋白。结论：经高浓度药物G418快速筛选的高拷贝整合酵母SMD1168细胞株，可表达人sCR1融合蛋白，并有与人体天然蛋白相同的抗原性。 [关键词]：可溶性1型补体受体；酵母SMD1168细胞；G418筛选 [中国分类号] R392.11 [文献标识码] A 收稿日期：2012－05－14；接收日期：2012－05－ 基金项目：河南省重点科技攻关项目（082102310046） 作者简介：周明武（1965-），男，安徽砀山人，主任医师，硕士； 电话E-mail：zmw153@126.com. *[通讯作者] 王广兰，电话：1394908886 E-mail：herrwangl@ Expression of human sCR1 in ferment cell SMD1168 screened by G418 ZHOU MingWu 1, ZHANG Changyuan 2, LIU Yali 3，WANG Gua-lan 4, LIU Liyuan 4, CANG Bao-cheng4, PEI pei4, ZHANG Lingyun 5. (1: The Orthopaedic Trauma Center of PLA, 2: Neurosurgical department of PLA 153rd Hospital, 3: Cardia-kidney medical department of PLA 153rd Hospital, 4: Center Laboratory medicine of Jinan Command PLA 153rd Hospital ,5: Military area outpatient of HENAN province, Zhengzhou 450042, China) [Abstract] Objective: Excretion vector pPIC9K carrying human complement receptor type 1(Scr1) gene was constructed and expressed in ferment cell SMD1168 as screened by G418. The rapid screening and assessment system were set up for clinical using in the future. Methords: Total human RNA was extracted from peripheral blood. The full length cDNA of human sCR1 gene was obtained by RT-PCR,constructed and cloned into Pichia pastoris SMD1168.Then the recombinant plasmid pPIC9K-sCR1 containing human sCR1was chosen by G418 resistance, and rapidly screened for its high copy carrier. Results: The obtained Pichia pastoris secretion type yeast carrier pPIC9K-sCR1 was chosen by G418, identified by PCR and Western blotting technique. We got a highly copied and integral recombinant ferment cell line. The